Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 https://doi.org/10.1186/s13052-019-0687-x

RESEARCH Open Access Isolation and identification of spp. From bronchoalveolar lavage of cystic fibrosis patients in Iran Mohammad Tabatabaei1*, Mahdi Dastbarsar1 and Mohammad Ashkan Moslehi2

Abstract Background: Pandoraea species are gram negative, motile, non-spore forming, rod shaped and oxidase positive, obligate aerobes , and have one polar flagellum. Most of Pandoraea species are associated with lung infections in cystic fibrosis patients. Cystic fibrosis is the most prevalent autosomal recessive hereditary disease in the world that affects various organs of the body. The main important cause of death in these patients is lung involvement. This study was conducted to isolate and identify Pandoraea bacterium from bronchoalveolar lavage and sputum samples of cystic fibrosis patients in Shiraz, Iran. Methods: In this research 31 samples of bronchoalveolar lavage and sputum were examined by culture and PCR method. Then confirmed isolates were evaluated for susceptibility to different antibiotics and ability to produce biofilm. Results: The results of this study after cultivation, purification and DNA extraction led to the isolation of 4 Pandoraea bacterium by PCR using specific primers. Antibiotic susceptibility test were indicated all isolates were resistant to gentamicin, amikacin and imipenem and susceptible to ciprofloxacin, trimethoprim-sulfumethoxazole, piperacillin and tetracycline. Ability to create biofilm was indicated by some of Pandoraea isolates. According to findings of this study, ability to synthesis biofilm by Pandoraea isolates and resistance to some antibiotics are very important. Conclusions: Our study notes the role of P. pnomenusa as an emerging pathogen that can cause chronic lung colonization in CF patients. Identification tools need to be accurate and must be based on molecular techniques. Also our findings should raise awareness about antibiotic resistance in cystic fibrosis patients in Iran and ability of including bacterial agents to produce biofilm is an alarm for public health. Thus clinicians should exercise caution about finding of clinical relevance of this pathogen to the infection and prescribing antibiotics, especially in cases of children infections. Keywords: Pandoraea spp., Cystic fibrosis, Bronchoalveolar lavage, PCR, Iran

Introduction faecigallinarum, P. vervacti, P. thiooxidance and four un- The genus Pandoraea is composed of aerobic, non-spore- named genomospecies. The majority of isolates have been forming, non-nitrate reducing, non-lactose fermenting, and isolated from respiratory samples from patients with cystic gram negative rods shaped bacteria, with polar flagella. fibrosis (CF) or other underlying chronic lung disease but They have been grown at 30–37 °C in 0.5 and 1.5% NaCl can also be isolated from other clinical samples (including or on Drigalski agar, assimilate D-gluconate, L-malate, and blood) and from the environmental samples such as soil, phenylacetate, and have , acid and alkaline phos- food, sea, and drinking water [1–5]. Its closest phylogenetic phatase and leucine arylamidase activity [1]. Different spe- relative is the genus Burkholderia and, like members of the cies of Pandoraea have been described: P. apista, P. genus Burkholderia,thePandoraea spp. are emerging im- norimbergensis, P. pnomenusa, P. pulmonicola, P. spu- portant respiratory pathogens, particularly in people with torum, P. fibrosis, P. terrae, P. oxalativorance, P. cystic fibrosis (CF). Species in this genus are often misiden- tified as Burkholderia cepacia complex (Bcc) or Ralstonia * Correspondence: [email protected] species owing to overlapping biochemical profiles without 1Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran differences that reliably distinguish between species [6]. It Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 2 of 8

is because of these limitations that reliable identification re- Whether Pandoraea spp. is truly pathogenic is not yet quires 16S ribosomal DNA sequence analysis. fully understood, there is evidence that infection with Pan- Cystic fibrosis is caused by mutations in the CFTR (cys- doraea spp. invokes a host inflammatory response. A num- tic fibrosis transmembrane conductance regulator) gene ber of virulence factors have been associated with clinical [7]. The commonest mutation is the deletion of phenyl- virulence of Pandoraea spp. and Bcc, including stimulation alanine at codon 508 (phe508del). This occurs in about of pro-inflammatory cytokine secretion known to cause 70% of patients with cystic fibrosis. The primary function lung tissue damage (IL-6 and IL-8), biofilm formation and, of the CFTR protein is as an ion channel that regulates li- in the case of Bcc, the ability to invade lung epithelial cells, quid volume on epithelial surfaces through chloride secre- which may contribute to the persistence of the strains in tion and inhibition of sodium absorption. The commonly the CF lung [16–20]. Indeed, biofilm formation by Pandor- accepted explanation for airway disease in cystic fibrosis is aea spp. and Bcc has been specifically associated with in- the “low volume” hypothesis. A reduced volume of airway creased resistance to antibiotics and maintenance of the surface liquid causes failure of mucociliary clearance, the bacteria in the lung [21–24]. Here, we report the results of lungs’ innate defense mechanism [8]. The mucociliary dys- isolation and identification of Pandoraea spp. and their function means that a patient with CF cannot effectively ability to form biofilms in vitro, in order to gain insight into clear inhaled bacteria. In addition, there is an excessive in- their virulence potential in CF lung infections. flammatory response to pathogens. For a given bacterial In the clinical microbiology laboratory, identification to load, a person with CF will have up to 10 times more in- the species level and differentiation of Pandoraea species flammation than a person with a lower respiratory tract from organisms belonging to the Bcc, R. pickettii,orR. infection but without the disease. The reasons for the ex- paucula may be problematic [1, 6, 25]. To aid in the iden- cessive inflammatory response to pathogens are not fully tification of these organisms, we used PCR-based identifi- understood. The abnormal composition and secretion of cation strategies based on the 16S rRNA gene (rDNA). mucus may also be important. At birth, the airway is unin- fected and probably uninflamed [9], but the end result of Materials and method the abnormalities described above is irreversible airway Sample collection damage with bronchiectasis and respiratory failure in most From November 2017 to August 2018, following the sed- patients. Ion and water abnormalities may also cause dis- ation of CF patients in Namazi hospital (Shiraz, Iran), 31 ease in other epithelia-lined organs. samples of bronchoalveolar lavage and sputum were col- The main source of morbidity and mortality in CF pa- lected. Then the samples were transferred to the bacteri- tients, is the decline in the pulmonary function subsequent ology laboratory, faculty of veterinary medicine of Shiraz to pathogenic colonization with non-fermenting Gram University, in cold condition. The study was approved by negative bacteria (NFGNB) that they encounter throughout the Ethics Committee of Shiraz University of Medical their lives.CF patients are particularly susceptible to infec- Sciences. tions caused by specific bacterial pathogens such as Pseudo- monas aeruginosa, Staphylococcus aureus and Haemophilus Culture and isolation influenzae [10]. To obtain strains and to compare culture and PCR re- Although Pandoraea species have also been isolated sults, cultures were performed initially as enrichment from sputum samples of CF patients, there is still very cultures. Upon arrival, bronchoalveolar lavage and spu- little known about their mechanisms of pathogenicity or tum were cultured in BHI broth and incubated aerobic- their roles in CF lung disease [11]. In addition, Pandor- ally at 37 °C for 24 h. Initial enriched cultures were aea isolates have been recovered from both CF and non- transferred to the enrichment culture plates by serial CF patients from a variety of clinical samples including streaking the cultures on sheep blood agar and incu- blood, sputum, urine, the upper airways and lung tissue bated aerobically at 37 °C for 24–48 h. [12]. The recovery of Pandoraea isolates from the blood Then, a loop full of the identified colonies were streaked of patients indicates that this organism is capable of in- on a new blood agar and incubated aerobically at 37 °C for vading tissue [13, 14]. Antibiotic therapy for treatment 24 h. Next, from culture positive plates, typical colonies of infection is difficult due to the limited number of were subjected to gram’s staining to study staining reac- antibiotics to which these species are susceptible: tions and cellular morphology under light microscope. tetracycline, imipenem and trimethoprim–sulfameth- Mixed and gram-negative bacteria were further sub cul- oxazole [15]. tured with due care, on both blood agar and MacConkey However, the clinical significance of colonization with agar plates for final identification. The growth of typical these organisms remains unclear [13] and there are limited colonies on both blood agar and MacConkey agar was and conflicting data available on the clinical outcome of characterized using blood agar for the presence and type patients colonized with Pandoraea. of haemolysis, and the general appearance of colonies Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 3 of 8

(morphology, color, shape, size and consistency) and the that used to extract DNA, and from the amplification and MacConkey agar for the ability to ferment lactose. Pure post-PCR room, in order to minimize contamination. A cultures of single colony type were further analyzed by negative control consisting of all component of reaction catalase and oxidase tests. Confirmation of bacteria to spe- mixture except the DNA template was included in all PCR. cies level was aided by using the biochemical tests Positive controls were included in the PCR from colonies including, metabolism of sugars such as glucose, fructose, that were confirmed according to NCBI gene bank sequen- lactose and tests for metabolic end products such as cing. In order to evaluate the PCR results electrophoresis in nitrate reduction, unease and citrate activity, growth on agarose gel was carried out. PCR amplicons were assessed cetrimide agarat 42 °C and O/F medium following stand- by loading 8 μlofthePCRproductplus2μlofloadingbuf- ard procedures. For final confirmation, isolates evaluated fer into separate wells of a 1% (w/v) agarose gel (Agarose I; by two PCR reactions. A first PCR assay was designed to Cinnagen, Iran) containing safe stain. A molecular weight identify all Pandoraea spp. Subsequent PCR assays were marker (50 bp; Cinnagen, Iran) was loaded into the first designed to identify individual Pandoraea species. well to determine the size of the amplified fragments. The gel was immersed in TBE buffer and subjected to a voltage DNA extraction difference of 100 V that led to separation of the fragments. A few colonies from the phenotypically characterized pure Visualization was undertaken using an ultraviolet transillu- cultures of Pandoraea from 24 to 48 h growth on blood minator (BTS-20, Japan), and the resulting image was cap- agar plates were transferred into 1.5 ml Eppendorf tubes. tured by a computer software program (Alpha Ease; Alpha Total DNA was prepared using the Gram negative DNA Innotech). extraction kit (Cinagen, Iran). The protocol for Gram For the final confirmation of isolates of Pandoraea, negative bacteria as described in the kit was followed for PCR products of Pan primers for some samples accord- extractions. The extracted DNA were determined to be of ing to the Macrogene Company’s instructions were pre- good quality and DNA concentration was measured using pared at 50 μl volume and were transferred for Nanodrop (10,000 V 3.52). DNA concentrations were sequencing. In addition, 16S rRNA gene sequencing for − adjusted to 15 ng μL 1 before PCR amplification. Finally, species identification was applied for some of the Pan- extracted DNA stored at −20 °C for further use. doraea isolates. The sequences were compared with pre- viously published 16SrRNA (GenBank accession no.NR- Master mixture of PCR 152002), Pandoraea gene sequences. The oligonucleotide primers usedinthisstudyweresynthe- sized by Cinagen Company (Iran). The forward and the re- Antimicrobial susceptibility testing verse primers sequences are as PanF (5´GGGCTYAACC Antimicrobial susceptibility testing for Pandoraea spp. iso- TGGGAACTGCATTC3´), and PanR (5´CGRYTTGGCRR lates was performed by disk diffusion method according to CCCTCTGTACCG3´) [26]. Also PCR evaluations have the Clinical and Laboratory Standards Institute (CLSI) [27]. been done with two universal primers for amplification of Disk diffusion method was performed on Muller-Hinton 16S rRNA gene. The reaction mixture solution for PCR was agar (Merck, Germany), using an inoculum of 105 CFU/ml. prepared using 2 μl of 10x PCR buffer, 1.5 μl(1.5mM) Antibiotic disks (Padtan teb, Iran) including amikacin, cip- MgCl2,1.2μl (200 mM) dNTPs, 1.2 μl (100 nmol) each pri- rofloxacin, trimethoprim- sulfumethoxazole, gentamicin, mer of Pandoraea,0.2UofTaq DNA polymerase, 14.7 μl piperacillin tetracycline and imipenem. These antibiotic H2O, and 3 μl of DNA template to have a final volume of disks were then placed on agar plates and incubated at 37 25 μl. Using 0.2 ml thin wall microtubes. After initial de- °C for 24 h. Escherichia coli (ATCC 25922) and Pseudo- naturation (at 94 °C for 5 min), amplification conditions monas aeruginosa (ATCC 27853) were used as quality were, denaturation at 94 °C for 1 min, annealing at 60 °C controls strain in each susceptibility determination. The for45s,andextensionat72°Cfor1min.Thiswasre- diameter of inhibition zone was measured in millimeters peated for 30 cycles in a Block assembly 96G thermocycler and isolates were scored as susceptible or resistant by com- with a hot top assembly (Analytic Jena, Germany), with a paring results with values recommended on standard final extension of 72 °C for 10 min and the PCR products charts. remained in the thermal cycler at 4 °C until they were col- lected. All DNA isolation procedures were carried out in a Biofilm assay Class II biological safety cabinet in a room geographically In order to evaluate the ability to create biofilms in Pan- separate from that used to set up reaction mixes and also doraea spp. isolates used the method of Merritt et al. from the ‘post PCR’ room, in order to prevent crossover and Wakimoto model [28, 29]. Briefly, 100 μl of each di- contamination by extraneous nucleic acids and in accord- luted culture was relocate into individual wells of micro- ance with good molecular diagnostic practice. All reaction titer dish and grown at 37 °C overnight. All wells were mixes were set up in a PCR hood in a room separate from stained with 125 μl of 0.2% (w/v) crystal violet solution Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 4 of 8

for 10 min at room temperature. The excess stain was Discussion washed twice with distilled water and the plate was Cystic fibrosis (CF) is the most common life-limiting allowed to air dried. 200 μl of 95% ethanol was added to autosomal recessive disease among people of European solubilize the stained biofilm and incubated for 10–15 heritage [30]. In the United States, about 30,000 individ- min at room temperature. 125 μl of each well was trans- uals have CF; most are diagnosed by six months of age. ferred into a new microtiter dish. Finally, the optical In Canada, about 4000 people have CF [31]. Around 1 in density (OD) of each sample was measured at 570 nm by 25 people of European descent, and one in 30 of Cauca- a spectrophotometer. The isolates with the optical dens- sian Americans [32], is a carrier of a CF mutation. Al- ity higher than 0.2 were considered as biofilm former though CF is less common in these groups, roughly one isolates. in 46 Hispanics, one in 65 Africans, and one in 90 Asians carry at least one abnormal CFTR gene [33, 34]. Ireland has the world’s highest prevalence of CF, at one Results in 1353 [35]. Identification of the bacterial species was made by obser- CF is a common genetic disease. Due to this condition, vation of their colonial morphology, gram staining reac- mucosal secretions in the lung can cause recurrence and tion and biochemical characteristics according to persistent respiratory infections. A number of opportun- Coenyeet al. (2000). Through culture 8isolates of Pan- istic pathogens settle in these patients. In most cases, in doraea with small gray circle colonies without haemoly- chronic infections, Pseudomonas, Burkholderia, as well sis was observed that were rod shapes in gram staining. as Pandoraea spp. have been isolated from the lung of Catalase and oxidase tests were positive [1]. CF patients, which play role in emerging pathogenesis. In this study, Pandoraea strains were isolated from clin- Pandoraea spp. are considered emerging pathogens in ical specimens, as in previous studies including bronchoal- the context of CF and are difficult to identify by conven- veolar lavage and sputum of CF patients. Also, most tional biochemical methods. Most Pandoraea spp. are isolates were involved in polymicrobial infections, including present in the lung of CF patients, lung infections, and Staphylococcus, Pseudomonas, Neisseria flavorans (accord- oral and dental infections. According to the studies con- ing to DNA sequencing), Delftia acidovorans (according to ducted to data on patients with CF and lung infection DNA sequencing) and Klebsiella. diseases, opportunistic pathogens that have been in- When these culture positive isolates evaluated by PCR, volved in causing diseases including Pseudomonas, Bur- using the stated primers, all the samples tested for the kholderia and Pandoraea species, have been isolated and presence of Pandoraea were identified as 100% positive identified [29, 36–38]. (Figs. 1, 2). One group of bacteria currently considered to be emerging CF pathogens belongs to the genus Pandoraea. The genus Pandoraea was described by Coenye et al. Antimicrobial susceptibility (2000) to differentiate them from other already well- In this study susceptibility to antibiotics was detected known CF pathogens, including Pseudomonas and two using 7 usual antibiotics prescribed for treatment of closely related Gram-negative rods, Burkholderia and chronic lung infection including amikacin, ciprofloxacin, Ralstonia species. In fact, phenotypic methods used by trimethoprim-sulfumethoxazole, gentamicin, piperacillin, many microbiology laboratories commonly lead to the tetracyline and imipenem. Antibiotic susceptibility test misidentification of Pandoraea species as either Burkhol- were indicated all isolates were resistant to gentamicin, deria or Ralstonia species [2, 4]. amikacin and imipenem and susceptible to ciprofloxacin, Pandoraea infection led to the production of high trimethoprim-sulfumethoxazole, piperacillin and tetra- levels of antibodies, and to a worsened CF lung disease cycline (Fig. 3). [4, 10, 39]. After first colonization, Pandoraea spp. were able to chronically colonize the CF respiratory tract (CFRT) [10 , 40–42], were transmissible between patients Biofilm assay [4, 37] and can produce severe lung diseases and Our target in this study was phenotypical survey of bio- bacteremia [12, 43]. The pathogenicity appears mainly film synthesis ability. According to tests carried out on supported by a pro-inflammatory response induction four Pandoraea isolates evaluated for the ability to cre- significantly greater than with P. aeruginosa [19, 44] and ate biofilms in LB, TSB and BHI medium, one isolate the treatment may be complicated by multidrug resist- create strong biofilm in BHI medium, one create strong ance conferred by carbapenem hydrolyzing oxacillinases biofilm in TSB medium, but all four isolate create weak [19]. The potential involvement of Pandoraea in com- biofilm in LB medium. This results indicate that biofilm plex interactions between microorganisms within the CF formation can differ according the culture media. airways was also suggested [44]. Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 5 of 8

In current study from 31 samples of bronchoalveo- In 2003, Jorgensen et al., examined the epidemic of P. lar lavage and sputum, by culture method eight Pan- apista in patients with cystic fibrosis. According to the re- doraea bacteria were isolated and identified. These sults of this study, it has been shown that P. apista should bacteria were isolated and purified using conventional be added to the ever increasing list of pathogens that can diagnostic methods such as culture and biochemical cause chronic lung infections in CF patients [43]. tests. But finally by PCR assay just 4 isolates con- In 2008, Karher et al. examined the pathogenic and firmed as Pandoraea. Also, in addition to Pandoraea genetic characteristics of Pandoraea species in lung spp. other bacteria including Staphylococcus, Pseudo- epithelial cells. In this study, 17 Pandoraea bacteria monas, Neisseria flavorans, Delftia acidovorans and were isolated including 5 species: P. apista, P. pnome- Klebsiella were also isolated. nusa, P.sputorum,P.pulmonicolaand P. norimber- In 2001, Coenye et al. identified and investigated spe- gensis [38]. cies of Pandoraea bacteria. In this study, from 123 sam- In a study conducted by Panickar and David (2015) ples, Pandoraea (69), Burkholderia (30), Ralstonia (9) from October 2012 to September 2014 on outpatient, and Pseudomonas aeruginosa (5) were isolated and iden- and lavage samples from 182 CF children treated at the tified. For the first time, PCR testing was performed to Royal Manchester Hospital, 5(3%) were Burkholderia, identify the member of genus Pandoraea, which were 17(19%) Exophiala, 32(18) Achromobacter and 32 (18%) able to detect P. apista, P. pnomenusa, P. sputorum, P. Rhodotorula, 18 (10%) Achromobacter,1Ralstonia and 6 pulmonicola and P. norimbergensis [26]. (3%) Pandoraea were identified. This study showed that Daneshvar and colleagues in 2001 examined the cel- the number of Pandoraea and Achromobacter bacteria lular effects of Pandoraea spp. on lung cells in CF in CF children is increasing [39]. patients. The study also found that it was possible to Antibiotic treatment of infections caused by Pandor- isolate the Pandoraea from the cultures of patient’s aea species is difficult because it has been demon- blood who did not have cystic fibrosis [2]. strated resistant against a wide range of antibiotics In the study of Spreet et al., conducted in Canada in such as ampicillin, cefazolin, piperacillin, azithroman, 2002, from a total of 447 CF patients in 8 provinces in- broad- spectrum cephalosporins and aminoglycosides, cluded 5 Pandoraea (1.1%), 412 Burkholderia (92.6%) and show a different response to quinolones, tri- and 5 Ralstonia (1.1%) bacteria, using culture tech- methoprim-sulfumethoxazole, colistin and carbapen- niques, biochemical tests and molecular techniques in- ems. Pandoraea species are resistant to most cluding PFGE, RAPD and RFLP were identified and antibiotics, including beta-lactams and aminoglyco- isolated [45]. sides. The bacteria in the subjects evaluated moderate sensitivity to imipenem, doxycycline and ceftriaxone, and an unusual antibiotic susceptibility pattern to car- bapenem was also found in Pandoraea bacteria and resistance to meropenem and sensitivity to imipenem has been reported. The result shows that for antibiotic treatment of Pandoraea infection, antibiogram test for each person has been done individually. Then if a person is allergic to imipenem they can use carbapenem. All Pandoraea species except P. apista, G9278 are resistant to amika- cin and cefazolin antibiotics, most of which are resist- ant to broad-spectrum cephalosporins, azithroman and piperacillin. Also, these organisms are resistant to aminoglycosides such as gentamicin, tobramycin and amikacin, but their susceptibility to fluroquinolones is different [40]. Puges et al. (2015), reported that P. sputorum was susceptible to imipenem and resistant to meropenem. Fig. 1 PCR amplification profile of Pandoraea spp. from DNA isolated This discrepancy has already been described by several directly from samples with PAN primers. Lane a: 50 bp DNA marker, authors and is because of meropenem-hydrolyzing β- Lane b: Negative control, Lane C: Positive control and Lanes D-G: lactamases. Some strains are resistant to both imipe- Positive samples (645 bp amplicon size). It should be noted that 4 nem and meropenem, and an imipenem-hydrolyzing samples were absolutely homologues with documented genomic oxacillinase named OXA-62 has been identified in P. sequencing of Pandoraea in NCBI genomic bank pnomenusa [45]. Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 6 of 8

Fig. 2 Genomic sequencing of Pandoraea documented in NCBI genomic bank

In this study susceptibility to antibiotics was ciprofloxacin, trimethoprim-sulfumethoxazole, tetra- detected using 5 antibiotics including amikacin, cycline and piperacillin. ciprofloxacin, trimethoprim-sulfumethoxazole, genta- Biofilms are microbial society encased in extracellular micin and piperacillin. Antibiotic susceptibility test polymeric substances (EPS) [18]. Biofilm formation sym- were indicated all isolates were resistant to gentami- bolizes a protective mode of growth that allows microor- cin, imipenem and amikacin and susceptible to ganisms to survive in hostile environments [46]. Biofilm

Fig. 3 Antibiogram results of Pandoraea isolates Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 7 of 8

is responsible for persistent in chronic infections, due to pulmonology and bronchoscopy provided bronchoalveolar lavage samples. their inherent resistance to antimicrobial agents. Bio- All authors have read and approved the final version of the manuscript. films are shown as being resistant to killing by a broad Funding range of antimicrobial agents [47]. Some bacteria such This research was funded by Shiraz University (Grant no. S9530963). as P. aeruginosa secrete the exopolysaccharide alginate during infection of the respiratory tract of individuals Availability of data and materials afflicted with cystic fibrosis and chronic obstructive pul- Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. monary disease [48]. Biofilm production has been con- sidered to be an important determinant of pathogenicity Ethics approval and consent to participate in most infections. In this study all 3 evaluated isolates Not applicable. showed strong biofilm formation ability. Consent for publication Not applicable. Conclusions In conclusionsthe results of our study indicate that Pan- Competing interests doraea species can be isolated from bronchoalveolar lav- The authors declare that they have no competing interests. age and sputum cultures of CF patients. The correct Author details identification of this bacterial species presents a chal- 1Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran. 2Shiraz lenge for diagnostic microbiology laboratories. Our study University of Medical Sciences, Shiraz, Iran. supports the use of genotypic methods to augment rou- Received: 20 April 2019 Accepted: 23 July 2019 tine phenotypic evaluation. The combined use of the two PCR assays described will allow the identification of References most Pandoraea species encountered in bronchoalveolar 1. Coenye T, Falsen E, Hoste B, Ohle’n M, Goris J, JRW G, et al. Description of lavage and sputum cultures of CF patients. Most import- Pandoraea gen. Nov. with sp. nov., antly, the use of these assays will substantially reduce sp. nov., sp. nov., sp. nov.and comb. nov. Int J Syst Evol Microbiol. 2000;50:887–99. the misidentification of Pandoraea spp. as Bcc. These 2. Daneshvar MI, Hollis DG, Steigerwalt AG, Whitney AM, Spangler L, Douglas tests will be a valuable adjunct in the evaluation of CF MP, et al. Assignment of CDC weak oxidizer group 2 (WO-2) to the genus bronchoalveolar lavage and sputum culture isolates and Pandoraea and characterization of three new Pandoraea genomospecies. J Clin Microbiol. 2001;39:1819–26. will allow more precise study of the prevalence and nat- 3. See-Too WS, Ambrose M, Malley R, Ee R, Mulcahy E, Manche E, et al. ural history of human infection by this emerging patho- Pandoraea fibrosis sp. nov., a novel Pandoraea species isolated from clinical gen. Finally our findings should raise awareness about respiratory samples. Int J Syst Evol Microbiol. 2019;69(3):645–51. https://doi. org/10.1099/ijsem.0.003147. antibiotic resistance in CF patients in Iran and their abil- 4. Degand N, Lotte R, Le Butor CD, Segonds C, Thouverez M, Ferroni A, et al. ity to produce biofilm is an alarm for public health. Thus Epidemic spread of Pandoraea pulmonicolain a cystic fibrosis center. BMC clinicians should exercise caution in prescribing antibi- Infect Dis. 2015;15:583. https://doi.org/10.1186/s12879-015-1327-8. 5. Jeong SE, Lee HJ, Jia B, Jeon CO. Pandoraea terrae sp. nov., isolated from otics, especially in cases of children infections. forest soil, and emended description of the genus Pandoraea Coenye et al. 2000. Int J Syst Evol Microbiol. 2016;66(9):3524–30. https://doi.org/10.1099/ Abbreviations ijsem.0.001229. ATCC: American type culture collection; Bcc: Burkholderia cepacia complex; 6. Henry DA, Mahenthiralingam E, Vandamme P, Coenye T, Speert DP. BHI: Brain heart infusion; bp: base pair; CF: Cystic fibrosis; CFTR: Cystic fibrosis Phenotypic methods for determining genomovar status of the Burkholderia transmembrane conductance regulator; CFU/ml: Colony forming units/ cepacia complex. J Clin Microbiol. 2001;39:1073–8. milliliter; CLSI: Clinical and Laboratory Standards Institute; 7. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. DNA: Deoxyribonucleic acid; dNTPs: Deoxyribonucleic triphosphates; Identification of the cystic fibrosis gene: cloning and characterization of EPS: extracellular polymeric substances; LB: Luria bertoni; ml: Milliliter; complementary DNA. Science. 1989;245:1066–73. μ NCBI: National Center for Biotechnology Information; ng L-1: nanogram/ 8. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, et al. Evidence microliter; nm: nanometer; O/F: Oxiation/ Fermentation; OD: optical density; for periciliary liquid layer depletion, not abnormal ion composition, in the PCR: Polymerase chain reaction; phe508del: Phenylalanine 508 Deletion; pathogenesis of cystic fibrosis airways disease. Cell. 1998;95:1005–15. rDNA: ribosomal Deoxyribonucleic Acid; rRNA: ribosomal ribonucleic Acid; 9. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of Taq: Thermus aquiticus; TBE: Tris Borate EDTA (Ethylenediaminetetraacetic pulmonary infections in cystic fibrosis. Am J RespirCrit Care Med. 2003;168: acid); TSB: Triptic soy broth; w/v: Weight/Volume 918–51. 10. Martina PF, Martínez M, Frada G, Alvarez F, Leguizamón L, Prieto C, et al. Acknowledgements First time identification of Pandoraea sputorum from a patient with cystic The authors wish to thank the staff member of department of pathobiology, fibrosis in Argentina: a case report. BMC Pulmon Medici. 2017;17:33. https:// and all of the people who collaborated with the authors, particularly doi.org/10.1186/s12890-017-0373-y. the laboratories of microbiology and biotechnology (Mr. M. Sorbi and S. 11. LiPuma JJ. Burkholderia and emerging pathogens in cystic fibrosis. Semin Golvagoei), faculty of veterinary medicine, Shiraz University. Respir Crit Care Med. 2003;24:681–92. 12. Pimentel JD, MacLeod C. Misidentification of Pandoraea sputorum isolated Authors’ contributions from sputum of a patient with cystic fibrosis and review of Pandoraea This study is the major component of the work toward the M.Sc. thesis of species infections in transplant patients. J Clin Microbiol. 2008;46:3165–8. the MD. MT as supervisor of this work provided guidance during the entire 13. Atkinson RM, Lipuma JJ, Rosenbluth DB, Dunne WM Jr. Chronic colonization experiment. MD wrote the first draft of the manuscript. Then, the whole with Pandoraea apista in cystic fibrosis patients determined by repetitive- manuscript critically revised by MT. MAM as an expert in pediatrics element-sequence PCR. J Clin Microbiol. 2006;44:833–6. Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 8 of 8

14. Stryjewski ME, LiPuma JJ, Messier RH Jr, Reller LB, Alexander BD. Sepsis, 40. Fernández-Olmos A, Morosini MI, Lamas A, García-Castillo M, García-García L, multiple organ failure, and death due to Pandoraea pnomenusa infection Cantón R, et al. Clinical and microbiological features of a cystic fibrosis after lung transplantation. J Clin Microbiol. 2003;41:2255–7. patient chronically colonized with Pandoraea sputorum identified by 15. Schneider I, Queenan AM, Bauernfeind A. Novel carbapenem-hydrolyzing combining 16S rRNA sequencing and matrix-assisted laser desorption oxacillinase OXA-62 from Pandoraea pnomenusa. Antimicrob Agents ionization-time of flight mass spectrometry. J Clin Microbiol. 2012;50:1096–8. Chemother. 2006;50:1330–5. https://doi.org/10.1128/JCM.05730-11. 16. Burns JL, Jonas M, Chi EY, Clark DK, Berger A, Griffith A. Invasion of 41. Kokcha S, Bittar F, Reynaud-Gaubert M, Mely L, Gomez C, Gaubert JY, et al. respiratory epithelial cells by Burkholderia (Pseudomonas) cepacia. Infect Pandoraeapulmonicola chronic colonization in a cystic fibrosis patient, Immun. 1996;64:4054–9. France. New Microbes New Infect. 2013;1:27–9. https://doi.org/10.1002/2 17. Cieri MV, Mayer-Hamblett N, Griffith A, Burns JL. Correlation between an in 052-2975.16. vitro invasion assay and a murine model of Burkholderia cepacia lung 42. Puges M, Debelleix S, Fayon M, Megraud F, Fayon M, Megraud F, et al. infection. Infect Immun. 2002;70:108–6. Persistent infection because of Pandoraea sputorum in a young cystic 18. Duff C, Murphy PG, Callaghan M, McClean S. Differences in invasion and fibrosis patient resistant to antimicrobial treatment. Pediatr Infect Dis J. translocation of Burkholderia cepacia complex species in polarized lung 2015;34(10):1135–7. https://doi.org/10.1097/INF.0000000000000843. epithelial cells in vitro. Microb Pathog. 2006;4:183–92. 43. Jørgensen IM, Johansen HK, Frederiksen B, Pressler T, Hansen A, Vandamme P, 19. Caraher E, Collins J, Herbert G, Philip G, Murphy PG, Gallagher CG, Crowe Koch C. Epidemic spread of Pandoraea apista, a new pathogen causing severe MJ, et al. Evaluation of in vitro virulence characteristics of the genus lung disease in cystic fibrosis patients. Pediatr Pulmonol. 2003;36(5):439–46. Pandoraea in lung epithelial cells. J Med Microbiol. 2008;57(Pt 1):15–20. 44. Costello A, Herbert G, Fabunmi L, Schaffer K, Kavanagh KA, Caraher EM, et 20. Green H, Jones AM. Emerging gram-negative bacteria: pathogenic or al. Virulence of an emerging respiratory pathogen, genus Pandoraea, in vivo innocent bystanders. Curr Opin Pulm Med, 2018; 24:000–000. https://doi. and its interactions with lung epithelial cells. J Med Microbiol. 2011;60:289– org/10.1097/MCP.0000000000000517. 99. https://doi.org/10.1099/jmm.0.022657-0. 21. CaraherE,DuffC,MullenT,McKeonS,MurphyP,CallaghanM,McCleanS. 45. Speert DP, Henry D, Vandamme P, Corey M, Mahenthiralingam E. Invasion and biofilm formation of Burkholderia dolosa is comparable with Epidemiology of Burkholderia cepacia complex in patients with cystic Burkholderia cenocepacia and Burkholderia multivorans.JCystFibros.2007;6:49–56. fibrosis, Canada. Emerg Infect Dis. 2002;8(2):181. 22. Chernish RN, Aaron SD. Approach to resistant gram negative bacterial 46. Caraher E, Collins J, Herbert G, Murphy PG, Gallagher CG, Crowe MJ, pulmonary infections in patients with cystic fibrosis. Curr Opin Pulm Med. McClean S. Evaluation of in vitro virulence characteristics of the genus 2003;9:509–15. Pandoraea in lung epithelial cells. J Med Microbiol. 2008;57(1):15–20. 23. Hoiby N, Krogh Johansen H, Moser C, Song Z, Ciofu O, Kharazmi A. 47. Davies D, Parsek M, Pearson J, Iglewski B, Costerton J, Greenberg E. The Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of involvement of cell-to-cell signals in the development of a bacterial biofilm. growth. Microbes Infect. 2001;3:23–35. Science. 1998;280:295–8. 24. Yu H, Head NE. Persistent infections and immunity in cystic fibrosis. Front 48. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause Biosci. 2002;7:d442–57. of persistent infections. Science. 1999;284:1318e1322. 25. Moore JE, Coenye T, Vandamme P, Elborn JS. First report of Pandoraea norimbergensis isolated from food-potential clinical significance. Food ’ – Publisher sNote Microbiol. 2001;18:113 4. Springer Nature remains neutral with regard to jurisdictional claims in 26. Coenye T, Liu L, Vandamme P, LiPuma JJ. Identification of Pandoraea species published maps and institutional affiliations. by 16S ribosomal DNA-based PCR assays. J Clin Microbiol. 2001;39:4452–5. 27. CLSI. M100-S25. Performance standards for antimicrobial susceptibility testing; Twenty-fifth informational supplement; 2015. 28. Merritt J, Kadour D, O'Toole G. Growing and analyzing static biofilms, Curr Protoc Microbiol. 01 2005; Unite1B.1. 29. Wakimoto N, Nishi J, Sheikh J, Nataro J, Sarantuya J, Iwashita M, Manago K, Tokuda K, Yoshinag M, Kawano Y. Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli. Am J Tro Med Hyg. 2004;71:687e690. 30. Edward T. Essential Medical Genetics, 2011. John Wiley & Sons. p. 312. ISBN 1-118-29370-3. Archived from the original on 2016-04-17. 31. The canadian facts & figures on cystic fibrosis. Archived from the original on 2013-06-16. 32. "Genetic Carrier Testing". Cystic Fibrosis Foundation. 2007. Archived from the original on 2010-03-23. 33. Rosenstein BJ, Cutting GR. The diagnosis of cystic fibrosis: a consensus statement. Cystic Fibrosis Foundation consensus panel. J Pediatr. 1998; 132(4):58995. https://doi.org/10.1016/S0022-3476(98)70344-0.PMID9580754. 34. Hamosh A, FitzSimmons SC, Macek M, Knowles MR, Rosenstein BJ, Cutting GR. Comparison of the clinical manifestations of cystic fibrosis in black and white patients. J Pediatr. 1998;132(2):255–9. https://doi.org/10.1016/S0022-34 76(98)70441-X.PMID9506637. 35. Farrell P, Joffe S, Foley L, Canny GJ, Mayne P, Rosenberg M. Diagnosis of cystic fibrosis in the Republic of Ireland: epidemiology and costs. Ir Med J 2007;100 (8): 557–560. PMID 17955689. Archived from the original on 2013-12-03. 36. Karatan E, Watnick P. Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Mol Biol Rev. 2009;73:310–47. 37. Panickar J, David T. Prevalence of emerging pathogens in a large paediatric CF centre. J Cyst Fibros. 2015;14:S68. 38. Ambrose M, Malley RC, Warren SJC, Beggs SA, Swallow OFE, McEwan B, et al. Pandoraea pnomenusa isolated from an Australian patient with cystic fibrosis. Front Microbiol. 2016;7:692. https://doi.org/10.3389/fmicb.2016.00692. 39. Mahenthiralingam E. Emerging cystic fibrosis pathogens and the microbiome. Paediatr Respir Rev. 2014;15:13–5. https://doi.org/10.1016/j. prrv.2014.04.006.